CLUH maintains functional mitochondria and translation in motoneuronal axons and prevents peripheral neuropathy

Transporting and translating mRNAs in axons is crucial for neuronal viability. Local synthesis of nuclear-encoded mitochondrial proteins protects long-lived axonal mitochondria from damage; however, the regulatory factors involved are largely unknown. We show that CLUH, which binds mRNAs encoding mitochondrial proteins, prevents peripheral neuropathy and motor deficits in the mouse. CLUH is enriched in the growth cone of developing spinal motoneurons and is required for their growth. The lack of CLUH affects the abundance of target mRNAs and the corresponding mitochondrial proteins more prominently in axons, leading to ATP deficits in the growth cone. CLUH interacts with ribosomal subunits, translation initiation, and ribosome recycling components and preserves axonal translation. Overexpression of the ribosome recycling factor ABCE1 rescues the mRNA and translation defects, as well as the growth cone size, in CLUH-deficient motoneurons. Thus, we demonstrate a role for CLUH in mitochondrial quality control and translational regulation in axons, which is essential for their development and long-term integrity and function.

The PDF file includes: Figs. S1 to S9 Legends for movies S1 to S4 Legends for tables S1 to S3 Other Supplementary Material for this manuscript includes the following: Movies S1 to S4 Tables S1 to S3 Fig. S1.NKO mice display an impaired rotarod performance

Fig. S7. CLUH does not alter the transport of CLUH target mRNAs
(A) The MS2-MCP system.An "RNA construct" with the target mRNA and a 3' attached MS2V5 sequence (RNA-MS2) is co-expressed with a "reporter construct", coding for the MCP coating protein tagged with HaloTag as the reporter protein (MCP-Halo).After the RNA construct is transcribed, MS2 stem loops form, allowing MCP-Halo to bind.Addition of Haloligand enables the visualization of the mRNA.
(A) Western blots depicting CLUH levels in forebrain, cerebellum and spinal cord of WT and NKO mice aged 5 months and spinal cord of WT and NKO E13.5 embryos.(B, C) Latency time to fall from a rotarod apparatus for male (B) and female (C) mice of the indicated genotypes.Data represent mean ± SD of 6-11 mice per group.Statistical significance was determined by one-way ANOVA followed by Dunn's and Dunnett's multiple comparison tests.

Fig. S2 .
Fig. S2.NKO motoneurons do not die in vivo, but show signs of axonopathy (A) Quantification of the percentage of mitochondria with dilated cristae in axons of the tibial branch of the sciatic nerve of mice at 5 and 14 months of age.Data represent mean ± SD of 3-6 mice (55-219 mitochondria per mouse).Statistical significance was determined by one-way ANOVA followed by Dunnett's multiple comparison test.(B) Gene ontology biological process (GOBP) analysis of upregulated (up, red) and downregulated (bottom, blue) transcriptome changes in the sciatic nerves of 5 months old male NKO mice compared to WT mice.Analysis was done using the EnrichR webtool.(C) Anterior spinal cord stained for Choline Acetyltransferase (ChAT, green) in mice of indicated genotypes at 14 months of age.Bar, 50 µm.(D) Quantification of the number of motoneurons in experiments shown in (C).Data represent mean ± SD of 3-5 mice (4-6 sections per mouse).

Fig. S3 .
Fig. S3.Phenotypes of NKO motoneurons (A) Primary spinal motoneurons at DIV 10 stained with TAU (green) and DAPI (blue).Bar, 50 µm.(B) Quantification of the number of TAU + neurons in experiments as in (A).Data represent mean ± SD of 4-5 mice (20 fields per mouse).Statistical significance was determined by Mann-Whitney test.(C) Nuclei (DAPI, blue) and apoptotic nuclei stained using TUNEL assay (green) of primary motoneurons at DIV 10.Bar, 50 µm.(D) Quantification of apoptotic nuclei in experiments as in (C).Data represent mean ± SD of 4 mice (10 fields per mouse).Statistical significance was determined by Welch's t test.(E) Western blot depicting CLUH levels in

Fig. S4 .
Fig. S4.NKO mitochondria are comparable to WT mitochondria in respect to clustering, morphology and membrane potential (A) Primary motoneurons stained with TOM20 (red).Representative axons are straightened and magnified on the right of each panel.Bars, 5 µm.(B) Quantification of the density of mitochondria in axons in experiments shown in (A).Data represent mean ± SD of 4-5 cultures (24-35 axons per culture).(C) Quantification of the abundance of mitochondrial DNA (mtDNA) to nuclear DNA (nDNA).Data represent mean ± SD of 5 motoneuronal cultures.(D) Quantification of the morphology of mitochondria in axons in experiments shown in (A).Data represent mean ± SD of 4 cultures (28-30 axons per culture).(E) Quantification of the mitochondrial membrane potential of axons of primary motoneurons loaded with TMRM.Neurons were treated with oligomycin after 5 minutes and CCCP after 35 minutes.Data represent mean ± SD of 4 WT and 5 NKO cultures.

Fig. S5 .
Fig. S5.Overexpressed Cluh-mCherry localizes to somas, axons and GCs (A) Western blot depicting CLUH levels in WT or KO mouse embryonic fibroblasts transfected with msCluh-mCherry (ms, mouse).(B) Overexpression of CLUH-mCherry in a primary spinal motoneuron shows signal in the soma, in protrusions and the GCs (arrowheads).Bar, 20 µm.(C) Area of CLUH + signal in GCs in respect to distal axons from images of the experiment shown in Fig. 4G.Data represent the mean ± SD of 7 WT independent cultures (6-12 cells per culture).Statistical significance was determined by one-way ANOVA followed by Dunnett's multiple comparison test.(D) GCs of primary motoneurons transfected with msCluh-mCherry (red) and stained for proteins of the cytoskeleton (green).Ac-TUB, acetylated tubulin, Tyr-TUB, tyrosinated tubulin.Bar, 10 µm.(E) A primary motoneuron transfected with untagged human CLUH and stained with an anti-human CLUH antibody (green).The boxed area containing a GC

Fig. S6 .
Fig. S6.Inhibition of ATP synthase induces the collapse of WT GCs but not NKO GCs (A) GCs transfected with GFP (grey) were imaged before and after addition of oligomycin.Bar, 10 µm.(B) Quantification of the percentage of reduction of the area of GCs after oligomycin treatment (15 min after administration) in experiments as in (A).Data represent mean ± SD of 8-9 GCs from different mice.Statistical significance was determined by Welch's t test.
Figure created with Biorender.com.(B) Kymograph of Atp5a1, Mdh2 and ActB mRNAs in axons of primary motoneurons using the MS2-MCP system depicted in (A).The first frame of the recording is shown as a straightened segment in the image above the kymograph.Bar, 5 µm.(C) Time spent in motion relative to the total tracked time and using a speed cut-off of more than 0.1 µm/s in experiments as in (B).(D) Division in three different motion-types (directed, oscillatory, stationary) as defined by total and maximal lateral displacement over the whole track or (E) by the mean square displacement in the first 25% of the track (active, diffusing, confined) in experiments as in (B).Data represents the mean ± SD of 4 cultures (10-93 mRNA dots per culture).

Fig. S8 .
Fig. S8.Validation of the proteomics from Boyden chambers (A) Neuron (left panel) and axon (right panel) compartments of Boyden chambers.Primary motoneurons are stained with MAP2 (red) and TAU (green).Bar, 25 µm.(B) Ratio of the protein intensities of the neuron and the axon compartments per sample.Hnrnpa1, Lmnb1 and Xpo1 were considered as proteins enriched in nuclei, Canx and Calr in ER and Ergic1 in Golgi.(C, D) Principal component analysis of proteomics in the neuron (C) and axon (D) compartment.Each day of collection of samples is depicted by a different colour.(E) Volcano plot showing protein changes in NKO versus WT neuron compartments of Boyden chambers.Mitochondrial proteins

Fig. S9 .
Fig. S9.ABCE1, RPL14 and RPS8 are reduced in NKO axons (A) Primary spinal motoneurons stained for ABCE1, RPL14 or RPS8 (green) and β-TUBULIN III (red).Axons are straightened and magnified next to the field showing motoneurons.Bars, 10 µm.(B) Quantification of the intensity of the fluorescence of ABCE1 (upper left graph), RPL14 (upper right graph) and RPS8 (lower graph) in axons of experiments as in (A).Data represent the mean ± SD of 5-7 cultures (30-58 axons per culture).Statistical significance was determined by Welch's t test.(C) Single confocal plane of a GC transfected with msCluh-mCherry (red) and stained for ABCE1 (green).